The major objective of this proposal is to develop a live oral Vibrio cholerae vaccine using recombinant DNA techniques for the control of the diarrheal disease cholera. Fully virulent strains of V. cholerae which have been extensively characterized in human volunteer studies and demonstrated to produce solid immunity to the disease will be genetically altered through the use of recombinant DNA techniques to specifically render them nontoxinogenic. No other virulence factors or antigens that may be necessary for stimulation of immunity will altered by the genetic techniques to be employed. The candidate vaccine strains will then be evaluated for their ability to induce protective immunity in the Removable Intestinal Tie Adult Rabbit Diarrhea (RITARD) model which is an excellent model of acute, fatal dehydrating diarrhea due to V. cholerae. The specific objectives of this project are as follows: Continue cloning and sequencing studies of cholera toxin. 2) Prepare deletion mutants of the cloned cholera toxin subunits, specifically A-B- and A-B+ mutants. 3) Reintroduce these defective toxin genes into well characterized strains of V. cholerae including both Classical and El Tor biotypes and Inaba and Ogawa serotypes. 4) Replace the proficient chromosomal toxin genes in these strains with the A-B- and A-B+ mutant genes. 5) Study the role of the B subunit in colonization and immunogenicity by varying the gene copy number of the B subunit. 6) Evaluate the various strains and antigen combinations in the RITARD model for colonization, immune response and protection against subsequent infection. 7) Prepare gene "libraries" of the entire V. cholerae genome so that the clones of other virulence factors can be studied and manipulated. 8) Evaluate the protein antigens profuced by the candidate vaccine strains by use of "Western blots" and sera from volunteers with induced cholera.